CN113615638B

CN113615638B - Construction method and application of precocious sexual animal model

Info

Publication number: CN113615638B
Application number: CN202010382813.7A
Authority: CN
Inventors: 孙强; 刘真; 熊志奇; 李奎
Original assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Current assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2022-12-27
Anticipated expiration: 2040-05-08
Also published as: CN113615638A

Abstract

The invention provides a construction method of a sexual precocity animal model and application thereof, in particular to a modeling reagent, which comprises (a) Follicle Stimulating Hormone (FSH) or analogues thereof; (b) testosterone or an analogue thereof. The present invention uses in combination (a) Follicle Stimulating Hormone (FSH) or an analog thereof; (b) Testosterone or analogues thereof can effectively construct a sexual precocity animal model.

Description

Construction method and application of precocious sexual animal model

Technical Field

The invention relates to the technical field of biology, in particular to a construction method and application of an animal model with precocious puberty.

Background

Non-human primates such as cynomolgus monkeys, rhesus monkeys, etc. have been widely used for neurobiological studies, particularly human brain disease and cognitive studies. Current gene editing monkey models (transgenics, knockouts and knockins) mainly utilize viral infections and programmable nucleases (ZFNs, TALENs, CRISPR-cas 9); however, most of the founder monkeys (F0) obtained by these gene editing methods have chimerism. This means that the F0 animals are not a good model, and in particular experiments involving comparisons between and within groups will be less rigorous. The second generation animals (F1) inherited the same transgene or mutation and were more amenable to study. However, macaques have a long prepubertal phase, and male macaques usually take 4 years or more to sexually mature. The chimerism and the long time of sexual maturation make the genetically modified monkey model not widely applicable.

Shortening the breeding time of F1 monkeys is very important for establishing animal models using genetically modified monkeys.

However, studies on the acquisition of functional sperm and generation of F1 generation by early maturing male monkeys induced by exogenous factors have not been reported in detail.

Therefore, there is an urgent need in the art to develop animal experimental models capable of inducing prematurity.

Disclosure of Invention

The invention aims to provide an animal experiment model capable of inducing precocity.

The invention provides in a first aspect a moulding agent comprising:

(a) Follicle Stimulating Hormone (FSH) or an analog thereof;

(b) Testosterone or an analogue thereof.

In another preferred embodiment, the Follicle Stimulating Hormone (FSH) or analog thereof is selected from the group consisting of: human Follicle Stimulating Hormone (FSH), a human follicle stimulating hormone analog, or a combination thereof.

In another preferred embodiment, the human FSH analog comprises monkey-derived FSH, goat-derived FSH, other animal-derived FSH, artificial recombinant FSH, urogonadotropin, human chorionic gonadotropin.

In another preferred embodiment, the testosterone or analogue thereof is selected from the group consisting of: testosterone, testosterone salts, or a combination thereof.

In another preferred embodiment, the testosterone salts comprise testosterone enanthate and testosterone propionate.

In another preferred embodiment, the molding agent is a liquid preparation.

In another preferred embodiment, the molding agent consists essentially (. Gtoreq.90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or entirely of (a) Follicle Stimulating Hormone (FSH) or an analog thereof and (b) testosterone or an analog thereof.

In another preferred embodiment, the concentration of component (a) in the molding agent is 1 IU/kg/day to 100 IU/kg/day, preferably 1 IU/kg/day to 20 IU/kg/day, more preferably 3IU/kg/day to 10 IU/kg/day.

In another preferred embodiment, the concentration of component (b) in the molding agent is 5-500 mg/week, preferably 50-250 mg/week, more preferably 100-150 mg/week.

In another preferred embodiment, the Follicle Stimulating Hormone (FSH) or analog thereof is derived from a mammal, more preferably from a rodent (e.g., mouse, rat), primate, and human.

In another preferred embodiment, the testosterone or an analogue thereof is derived from a mammal, more preferably from a rodent (e.g., mouse, rat), primate, and human.

In another preferred embodiment, the molding agent is an agent for preparing a non-human mammal animal model for inducing precocious puberty.

In another preferred embodiment, the precocious puberty comprises male precocious puberty.

In another preferred embodiment, the animal model comprises an animal model of a non-human mammal.

In another preferred embodiment, the animal model comprises an animal model that has not been genetically engineered or has been genetically engineered.

In another preferred example, the genetic engineering operation comprises ZFN (zinc-finger nuclei), TALEN or criprpr techniques.

In another preferred embodiment, the genetically engineered animal model comprises an animal model that is heterozygous for the knockout Prrt2 gene.

In another preferred embodiment, the non-human mammal comprises a non-human primate.

In another preferred embodiment, the non-human primate comprises a monkey, a chimpanzee.

In a second aspect, the invention provides the use of a modelling agent according to the first aspect of the invention in the manufacture of a medicament or formulation for inducing an animal model of non-human mammal sexual precocity.

In another preferred embodiment, the formulation is a liquid formulation.

The third aspect of the invention provides a method for preparing a sexual precocity animal model, which comprises the following steps:

(a) Providing a mammal;

(b) Administering a first component and a second component to said mammal, and culturing for a time T1, thereby obtaining said mammal's precocious animal model;

wherein the first component comprises Follicle Stimulating Hormone (FSH) or an analog thereof; the second component comprises testosterone or an analogue thereof.

In another preferred embodiment, said administration comprises injection, preferably intramuscular injection.

In another preferred embodiment, the first component and the second component are injected sequentially or simultaneously.

In another preferred embodiment, when the first component and the second component are injected sequentially, the interval between the injection of the first component and the injection of the second component is 1 to 60 days, preferably 5 to 30 days, more preferably 5 to 10 days.

In another preferred embodiment, T1 is between 30 days and 360 days, preferably between 60 days and 240 days, and more preferably between 90 days and 180 days.

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the concentration of said first component is between 1 IU/kg/day and 100 IU/kg/day, preferably between 1 IU/kg/day and 20 IU/kg/day, more preferably between 3IU/kg/day and 10 IU/kg/day.

In another preferred embodiment, the concentration of the second component is 5-500 mg/week, preferably 50-250 mg/week, more preferably 100-150 mg/week.

In a fourth aspect, the invention provides the use of an animal model prepared by the method of the third aspect of the invention as an animal model for studying the reduction in time to production of an animal model from a non-human mammal.

In a fifth aspect, the invention provides a non-human mammalian model prepared by a method according to the third aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows that the time to sperm production by male cynomolgus monkeys was successfully shortened by the combined use of FSH and testosterone.

FIG. 2 shows that the activity and fertilization rate of cynomolgus monkey sperm obtained by accelerated sexual maturation is comparable to that of normally sexually mature cynomolgus monkey sperm; the cynomolgus monkey sperm obtained by the accelerated sexual maturation can be used for constructing embryos and obtaining offspring cynomolgus monkeys.

FIG. 3 shows that the genome-wide methylation level of cynomolgus monkey sperm obtained by accelerated sexual maturation is comparable to that of a normally sexually mature cynomolgus monkey sperm.

FIG. 4 shows that the body growth curve of the offspring cynomolgus monkey using the cynomolgus monkey sperm obtained by accelerated sexual maturation is comparable to the body growth curve of the normal offspring cynomolgus monkey.

FIG. 5 shows that the intelligence development of the offspring cynomolgus monkey using the cynomolgus monkey sperm obtained by accelerated sexual maturation corresponds to the body growth curve of a normal offspring cynomolgus monkey.

FIG. 6 shows progeny PRRT2 knock-out cynomolgus monkeys using PRRT2 knock-out cynomolgus monkey sperm obtained from accelerated maturation.

Detailed Description

The present inventors have made extensive and intensive studies and have unexpectedly found that a sexual precocity animal model (particularly, a male sexual precocity animal model) can be efficiently constructed by using (a) Follicle Stimulating Hormone (FSH) or an analog thereof and (b) testosterone or an analog thereof in combination. Further, the present inventors have unexpectedly found that a sexual precocity animal model (particularly a male sexual precocity animal model) can be efficiently constructed by injecting (a) Follicle Stimulating Hormone (FSH) or an analog thereof and (b) testosterone or an analog thereof into a non-human mammal sequentially or simultaneously, and that the animal model of the present invention can be used for the research of shortening the production time of the animal model of the non-human mammal, thereby completing the present invention.

Prrt2

Prrt2 is a gene encoding a proline-rich transmembrane protein 2. Single gene mutation of Prrt2 results in loss of function, leading to paroxysmal dyskinesia.

Follicle Stimulating Hormone (FSH) or analogs thereof

FSH, a follicle stimulating hormone, is a hormone secreted by anterior pituitary basophils and is a glycoprotein. Follicle stimulating hormone regulates a series of physiological processes related to development, growth, adolescent sexual maturity and reproduction of a human body, and stimulates the maturation of germ cells. FSH internally divides at a rate later than other cytokines and primarily acts to promote follicular maturation. Promote the proliferation and differentiation of follicular granular layer cells and promote the growth and development of the whole ovary. When acting on the seminiferous tubules of testis, it can promote the formation of sperms.

In a preferred embodiment, follicle Stimulating Hormone (FSH) or an analog thereof includes, but is not limited to: follicle Stimulating Hormone (FSH), FSH analogs (e.g., monkey-derived FSH, goat-derived FSH, and other animal-derived or artificial recombinant FSH, urogonadotropin, human chorionic gonadotropin).

Testosterone or analogue thereof

Testosterone (also called testosterone, testosterone or testosterone) is a steroid hormone, which is secreted by male testis or female ovary, and adrenal gland also secretes a small amount of testosterone, and has the functions of maintaining muscle strength and quality, maintaining bone density and strength, refreshing and improving physical fitness.

In a preferred embodiment, testosterone or an analog thereof includes, but is not limited to: testosterone, testosterone salts (such as testosterone enanthate, testosterone propionate, and other testosterone salts).

Molding reagent

The present invention also provides a modeling agent comprising an effective amount of (a) Follicle Stimulating Hormone (FSH) or an analog thereof; and (b) testosterone or an analogue thereof.

Typically, (a) Follicle Stimulating Hormone (FSH) or an analog thereof; and (b) testosterone or an analogue thereof, in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is generally from about 5 to about 8, preferably from about 7 to about 8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that promotes precocious puberty (especially precocious puberty) in mammals after transfection, and does not cause death in the animal. In a preferred embodiment of the present invention, the effective amounts are: follicle Stimulating Hormone (FSH) or an analog thereof at 1 IU/kg/day to 100 IU/kg/day, preferably at 1 IU/kg/day to 20 IU/kg/day, more preferably at 3IU/kg/day to 10 IU/kg/day. Testosterone or an analogue thereof, 5-500 mg/week, preferably, 50-250 mg/week, more preferably, 100-150 mg/week. Preferably, the effective amount is administered in a single injection.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier, including for administration of a therapeutic agent. The pharmaceutically acceptable carrier which may be used in the present invention is not particularly limited and may be one or more compatible solid or liquid fillers or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with the adipose mesenchymal progenitor cells of the present invention without significantly diminishing their therapeutic effectiveness. Examples of pharmaceutically acceptable carrier moieties of the invention are physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions, suitable aqueous and nonaqueous carriers, diluents, solvents or excipients including water, ethanol, polyols and suitable mixtures thereof. In addition to the conventional vectors described above, optimized vectors can also be designed based on the properties of the adipose mesenchymal progenitor cells. The carrier is preferably an infusion solution carrier and/or an injection carrier.

The molding agent of the present invention comprises an effective amount of (a) Follicle Stimulating Hormone (FSH) or an analog thereof; and (b) testosterone or an analogue thereof. The pharmaceutical preparations should generally be adapted to the mode of administration, and the pharmaceutical molding agents of the present invention may be prepared in the form of injection preparations, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The molding agent of the invention can also be prepared into a sustained-release preparation.

The molding agent of the present invention is preferably an injectable preparation, more preferably an intramuscular injectable preparation. In another preferred embodiment, the Follicle Stimulating Hormone (FSH) or its analog is 1 IU/kg/day-100 IU/kg/day, preferably 1 IU/kg/day-20 IU/kg/day, more preferably 3 IU/kg/day-10 IU/kg/day. Testosterone or an analogue thereof, 5-500 mg/week, preferably, 50-250 mg/week, more preferably, 100-150 mg/week. The injection mode of the molding agent is not particularly limited, and the molding agent can be a single injection preparation or a combination of multiple injections. In a preferred embodiment of the present invention, the molding agent is a single injection.

In the invention, the molding agent is preferably an injection preparation, and more preferably an intramuscular injection preparation.

Animal model

In the present invention, a very effective non-human mammalian model of induced prematurity is provided.

In the present invention, the animal model includes an animal model which is not genetically engineered or genetically engineered.

In a preferred embodiment, the genetic engineering operation comprises ZFN (zinc-finger nuclei), TALEN or criprpr technology.

In a preferred embodiment, the genetically engineered animal model comprises an animal model that is heterozygous for a knockout Prrt2 gene.

In a preferred embodiment, the non-human mammal comprises a non-human primate.

In a preferred embodiment, the non-human primate comprises a monkey, a chimpanzee.

The main advantages of the invention include:

(a) The present invention has for the first time found that the combined use of (a) Follicle Stimulating Hormone (FSH) or an analog thereof and (b) testosterone or an analog thereof is effective in constructing an animal model that induces precocious puberty in a non-human mammal.

(b) The present invention has for the first time found that (a) Follicle Stimulating Hormone (FSH) or an analog thereof and (b) testosterone or an analog thereof, injected into a mammal either sequentially or simultaneously, are effective in constructing an animal model that induces sexual precocity in a non-human mammal, and that the animal model of the present invention can be used to shorten the time of production of the animal model in a non-human mammal (e.g., a non-human primate mammal) (e.g., by promoting sexual precocity, shortening the time between generations).

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials used in the examples are all commercially available products unless otherwise specified.

General procedure

The experimental methods include hormone injection, sperm collection, assisted reproduction, gene editing, HE staining, methylation sequencing, behavioral analysis, and the like.

Hormone injection: recipient experimental animals hFSH and testosterone were administered by intramuscular injection. Wherein FSH is injected at 3IU per kg per day and testosterone is injected at 125mg per week.

Collecting sperms: the collection of monkey sperms is carried out by a microcurrent penis stimulation method. The collected semen was liquefied at 37 ℃ for 15 minutes and then washed 2 times with TH3 for single sperm injection.

Taking eggs, injecting single sperms and transplanting embryos: the ovum-taking operation adopts a laparoscope negative pressure suction method. Single sperm injection was performed using a Piezo piezoelectric rupture membrane system. The embryo transplantation adopts a minimally invasive operation fallopian tube transplantation method.

Histological analysis: the testis tissue was fixed with Bouin fixative for 48 hours, and was stained with hematoxylin-eosin after sectioning.

And (3) sperm quality analysis: and analyzing sperm motility proportion and sperm straight-moving proportion by adopting sperm quality analysis of a CASA system.

DNA methylation analysis: DNA methylation analysis was performed by sulfite sequencing.

And (3) behavioral analysis: the method for detecting the intelligence development condition of the child cynomolgus monkey by adopting the classical wisconsin behavior test mainly comprises a black and white block test, a Hamilton test and a learning test.

Examples

The age of sexual maturation (production of motile sperm) of normal male monkeys was first studied in the monkey population. Semen was collected from 37 monkeys, including 8 monkeys at 36-42 months of age, 7 monkeys at 45-48 months of age, 17 monkeys at 50-55 months of age, and 5 monkeys at 59-66 months of age. In 15 monkeys between 36-42 months and 45-48 months, no semen or sperm was collected. However, 13/17 monkeys in the 50-55 month group and 4/5 monkeys in the 59-66 month group produced motile sperm (FIG. 1A). Thus, this suggests that 50 months should be a threshold for sexual maturation in male cynomolgus monkeys in the monkey population.

To accelerate the macaque sexual maturation process, 9 male young macaques (macaque) of 3 age groups (macaque group) (-0.5 year, n =2; -1,n =4; -2 years, n = 3) were intramuscularly injected with human follicle stimulating hormone (FSH, 3 IU/kg/day) and testosterone (testosterone enanthate, 125 mg/week), respectively; control was performed in 3 monkeys (aged about 1 year) without hormone injection. Monkey semen was collected by electrical stimulation of the penis and the collected semen was examined under an inverted microscope for the presence of sperm. After 4-6 months of hormone injection, motile sperm were detected in semen in 4/9 monkeys (FIG. 1B, C). Untreated 3 control monkeys produced no sperm. After 4-6 months of hormone injection, 4/9 of the monkeys detected motile sperm in the semen (fig. 1b, c); no sperm were produced in 3 monkeys of the control group.

4 monkeys produced mature sperm, and were from both the 1-year group (n =2,2/4) and the 2-year group (n =2,2/3), and the semi-year group (n =2,0/2) did not produce sperm (table 2).

The success rate of obtaining viable sperm in the first round of experiments was limited (4/9). In the second round of experiments, two age groups (-half year, n =2; -1 year, n = 3) were treated according to the same test method as in the first round. Another control group was 3 monkeys (aged about 1 year) injected with no hormone. Swimming sperm were obtained from 3 young monkeys aged 1 year old after 7 months, 11 months and 11 months of hormone treatment. The two monkeys, approximately half-year old, were treated with hormones for 8 months and 11 months, respectively, to obtain motile sperm. While sperm were not obtained in the control group. Thus, by extending the hormone treatment time, sperm from 5 hormone-treated monkeys were successfully collected (fig. 1D and table 2).

Monkeys were examined monthly and their weights and testicular volumes recorded for two runs. Mean body weight and testicular volume were significantly increased during hormone treatment compared to control group (fig. 1e, f and G). Testis tissue sections of the control group and the treated group were stained with hematoxylin-eosin. Compared to the immature testicular tissue of the age-matched control group, the hormone-treated monkeys had significantly increased vas deferens diameter and the presence of mature sperm (fig. 1H and I). Sperm motility and quality were then examined in 4 hormone-treated monkeys and 3 naturally mature (5 year old male) control monkeys using Computer Assisted Sperm Analysis (CASA) techniques. The rate of motile sperm in the 3 control monkeys was 94.7%, 88.7% and 64%, respectively. The corresponding sperm count (class a and class B) ratios were 86.8%, 70.9% and 37.2%, respectively. The sperm motility rates of 4 monkeys treated with hormone were 97.4%, 95.2%, 91.8%, and 63.9%, respectively. The corresponding sperm correspond to 90.9%, 86.3%, 77.6% and 36.4% respectively. Both the control group and the hormone-treated group had one monkey, which had low sperm motility and quality. Meanwhile, the ratio of motile sperm and sperm of the hormone-treated group was not significantly different from that of the control group, indicating that the sperm quality of the hormone-treated group was similar to that of the naturally mature control group (FIGS. 2A, B).

To assess the epigenetic status of sexually mature monkey sperm, 5-methylytosine (mC) genome-wide and single base resolution mapping was performed using bisultate-sequencing (BS-Seq) with a 15.4 fold average depth of naturally mature monkey (# 2) sperm sample DNA strands and a 13.6 fold average depth of prematured monkey (# 49). In both assays, approximately 70% of the genomic cytosines in each sample were covered at least 5 times. The mean methylation levels and densities of cytosines in both samples were analyzed and no significant difference was found between the hormone-treated and control groups (fig. 3, a-F).

Sperm from hormone-treated young monkeys were used to propagate offspring. Sperms of two hormone-treated monkeys were injected into oocytes of monkey MII stage by ICSI method, and the obtained embryos were transferred into synchronized gestational monkeys. For the 2 year old hormone treated group, sperm from monkey #49 (4 months after hormone treatment) were processed as described above. A total of 28 oocytes were injected to obtain 25 fertilized eggs, 24 of which were transplanted into 6 pregnant monkeys, and healthy offspring were obtained in 2 pregnancies (named HI 1 and HI 2, respectively; "hormone injection").

For the 1 year old hormone-treated group, sperm were obtained from 62# monkeys (after five months of hormone treatment) for ICSI,73 oocytes and 59 zygotes, 49 of which were transferred into 17 passages of pregnant monkeys, resulting in 8 pregnancies, 3 healthy monkeys (designated HI 3, HI 4, HI 5, FIGS. 2E-H) born, and remaining aborted or stillborn. The main causes of miscarriage may be double and triple gestation. In total, 109 oocytes were used for ICSI and 92 fertilized egg sperm were from hormone-treated young monkeys. The fertilization rate is 84.4%, and compared with the natural mature sperm, the fertilization rate has no obvious difference. The pregnancy rate was 40.7% (11/27), similar to that with natural mature sperm (FIG. 2C, D). In summary, we obtained 11 pregnant monkeys and 5 healthy offspring (table 1) using 2 hormone-treated young monkey sperms for ICSI, where table 1 describes fertilization rates and birth after single sperm injection using 1 year old, 2 year old, PRRT2 knockout monkeys induced mature sperm and normal natural mature sperm, respectively.

Parental source analysis was performed on 5 live HI monkeys using a Short Tandem Repeat (STR) genotyping method. Analysis of the 24 STR loci of 5F 1-generation live monkeys confirmed their paternal origin (table 3). Physical examination was performed on 5 HI monkey progeny obtained from young monkey sperm. Body weight, abdominal circumference, head circumference length, heart rate, respiratory rate and body temperature were recorded weekly after birth. No significant differences were found in 4 physiological parameters (body weight, abdominal circumference, head circumference and head and neck length) over a period of 23 months for 5 HI monkeys and 6 age-matched control monkeys (fig. 4, a-D). No abnormalities were found in other observed traits. Thus, hormone-treated monkey sperm were of the same quality as naturally mature monkey sperm in producing healthy offspring. The cognitive function of 5 HI monkeys was tested using a Wisconsin General Test Apparatus (WGTA). 5 age-matched naturally-born offspring were used as controls. Although there were considerable differences between the two groups, no significant differences were found between the two groups in terms of the adaptation, discrimination and reversal steps of the black/white test. In the subsequent Hamilton search test, both groups showed similar learning curves in a forced set-breaking step (FIG. 5A, B). Finally, a prize association learning test was performed by identifying the toy within 40 days of 240. The correct rate for both the HI group and the control group increased slowly with increasing training time (fig. 5C-H). The results show that, on the intelligence level, the offspring generated by the sperms of the monkeys induced by the hormones and the sperms of the naturally propagated monkeys have no obvious difference.

This hormone-induced sexual maturation approach was then applied to shorten the passage of genetically modified monkeys.

Prrt2 is a gene encoding a proline-rich transmembrane protein 2. Single gene mutation of Prrt2 results in loss of function, leading to paroxysmal dyskinesia. To accelerate the F1 passage, two 12-month old Prrt2 knockout F0 monkeys (designated P11 and P12, respectively) were used for hormonal treatment. After 7 and 12 months of hormone injection, we successfully obtained motile sperm from P12 and P11. Embryos were constructed from ICSI using sperm from these two prrt2 knockout F0 monkeys. The embryos cultured in vitro to the blastocyst stage were genotyped by PCR and DNA sequencing. The results indicated that all sequenced embryos were single gene mutations of the Prrt2 gene target sequence (figure 2i, k). 28 Prrt2F1 embryos containing P11 sperm were transplanted into 11 pregnant monkeys, resulting in 5 pregnant monkeys, 7 fetuses, and 4Prrt2F1 offspring survived (FIG. 2J, FIG. 6, A-D). Genetic analysis revealed that 7 fetuses, like the previously examined fetuses, all had single-gene mutations in the target sequence of the Prrt2 gene (table 4), where table 4 specifically describes the sex, date of birth, and mutation status of 4 surviving Prrt2F1 offspring. .

In the present invention, 100% efficiency of normally motile sperm was successfully obtained from WT male cynomolgus monkeys by precocity induction using testosterone and Follicle Stimulating Hormone (FSH) injection. Healthy F1 generations were bred with sperm from sexually premature monkeys, and these offspring had normal physical growth and cognitive development. Transgenic F1 monkeys can also be produced within two years using hormone-induced sexual precocity. Therefore, the method can be used as a common and simple method, shortens the generation time of the gene modified F1 monkey model, and contributes to the application of the non-human primate model in future scientific research.

TABLE 1 efficiency of FI monkey production

TABLE 2 sperm production in hormone-treated and control monkeys

TABLE 3 STR analysis of HI monkeys

TABLE 3 (continue)

Table 4 summary of Prrt2F1 monkeys

Note: in the present invention, the oocytes are oocytes from normal monkeys, and the different oocyte origins are numbered for oocytes from different monkeys.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A molding agent, characterized by being composed of:

(a) Follicle Stimulating Hormone (FSH) or an analog thereof;

(b) Testosterone or an analogue thereof, said modelling agent being an agent for the preparation of a model for inducing a juvenile non-human mammal sexual precocity animal, said Follicle Stimulating Hormone (FSH) or an analogue thereof being selected from the group consisting of: human Follicle Stimulating Hormone (FSH), human follicle stimulating hormone analogs, or combinations thereof; the testosterone or analogue thereof is selected from the group consisting of: testosterone, testosterone salts, or a combination thereof; the non-human mammal is a non-human primate;

administering said Follicle Stimulating Hormone (FSH) or analogue thereof to the young non-human mammal in an amount of 1 IU/kg/day to 100 IU/kg/day, and administering said testosterone or analogue thereof to the young non-human mammal in an amount of 5 to 500 mg/week.

2. The molding agent according to claim 1, wherein said precocious puberty comprises male precocious puberty.

3. Use of a molding agent according to claim 1 for the preparation of a medicament or formulation for inducing a young non-human mammal animal model of sexual precocity.

4. A method of making a young non-human mammal precocious animal model, comprising:

(a) Providing a non-human mammal;

(b) Administering to said mammal a first component and a second component, and culturing for a time T1, thereby obtaining said young non-human mammal precocious animal model;

wherein the first component is Follicle Stimulating Hormone (FSH) or an analog thereof; the second component is testosterone or analogues thereof;

the Follicle Stimulating Hormone (FSH) or analog thereof is selected from the group consisting of: human Follicle Stimulating Hormone (FSH), a human follicle stimulating hormone analog, or a combination thereof; the testosterone or analogue thereof is selected from the group consisting of: testosterone, testosterone salts, or a combination thereof;

the non-human mammal is a non-human primate;

the dosage of the first component is 1 IU/kg/day-100 IU/kg/day, and the dosage of the second component is 5-500 mg/week.

5. The method of claim 4, wherein the first component is present in an amount of from 1 IU/kg/day to 20 IU/kg/day.

6. The method of claim 5, wherein the first component is present in an amount of from 3IU/kg/day to 10 IU/kg/day.

7. The method of claim 4, wherein the second component is present in an amount of 50 to 250mg per week.

8. The method of claim 7, wherein the amount of the second component is from 100 to 150mg per week.

9. Use of an animal model prepared by the method of claim 4 as an animal model for studying the reduction of the time to production of an animal model from a non-human mammal.